Combination of the NS3/4A Protease Inhibitor ITMN-191 (R7227) with the Active Moiety of the NS5B Inhibitors R1626 or R7128 Enhances Replicon Clearance and Reduces the Emergence of Drug Resistant Variants
H. Tan1; S. Rajyaguru2; T. Wu2; M. McCown2; S. Ali2; W. Jiang2; M. J. Otto3; P. A. Furman3; I. Najera2; K. Klumpp2; J. Symons2; N. Cammack2; L. M. Blatt1; S. Seiwert1
1. Intermune, Inc, Brisbane, CA, USA.
2. Roche Palo Alto LLC, Palo Alto, CA, USA.
3. Pharmasset Inc., Princeton, NJ, USA.
Background: ITMN-191 is an inhibitor of HCV NS3/4A protease activity, and R1626 and R7128 are nucleoside inhibitors of the polymerase activity of HCV NS5B. All three compounds promote multi-log10 reductions in circulating HCV RNA in chronic HCV patients when administered for short durations as monotherapy. Here, to support future clinical studies that would combine ITMN-191 with R1626 or R7128, we investigated the combined antiviral effect of these compounds.
Methods: In the HCV clearance assay, cells harboring an HCV genotype 1b replicon were treated for 2 weeks with ITMN-191, the active moiety of R1626 (R1479), the active moiety of R7128 (PSI 6130), or a combination of inhibitors in the absence of G418 selection for replicon retention. Cells were counted and aliquots harvested for RT-PCR-based quantification of replicon RNA levels under G418 selection over 4 subsequent weeks. In the colony formation assay, cells were treated with either one or two compounds at 1X, 10X, or 15X their respective EC50. After 3 weeks in culture with G418, cells were fixed and stained with crystal violet or total cellular RNA extracted. For drug-drug interaction studies, HCV replicon cells were treated for 3 days with a pair of inhibitors in checkerboard dilution and percent reductions of reporter gene activity obtained.
Results: In the HCV clearance assay, 18 nM ITMN-191 and low μM concentrations of the active moiety of R1626 and R7128 (18 μM & 27 μM, respectively) eliminated HCV replicon in the absence of G418 selective pressure for replicon retention. Addition of the lowest tested concentration of ITMN-191 (6 nM) to the lowest tested concentration of the active moiety of R1626 or R7128 (0.3 μM & 0.45 μM, respectively) resulted in replicon clearance, demonstrating significant combined antiviral effect. In the colony formation assay in the presence of G418 selective pressure for replicon retention, NS5B inhibitors at 10X or 15X EC50 supported replicon clearance and did not result in drug resistant colonies. Similar treatment with ITMN-191 selected resistant colonies, but these were suppressed by an NS5B inhibitor at 1X EC50. Drug-drug interaction studies over a 3 day incubation period demonstrated additive to slightly synergistic interactions between the two inhibitor classes.
Conclusions: The combination of ITMN-191 with the active moeity of either R1626 or R7128 results in enhanced antiviral activity and suppression of ITMN-191 resistant variants. These findings suggest that the combination of ITMN-191 with R1626 or R7128 may confer significantly greater antiviral activity than has been observed with these agents in previous monotherapy trials.
